5×106 macrophages (approximate ratio 25:1). The interacting cells were incubated for 1 h at 37°C/5% CO2, in the presence or absence of 10% serum, washed, and incubated for 24 h with RPMI medium

and 10% serum. Supernatants were collected at 6 and 24 h. Interaction assays between human DC and iC3b-opsonized apoptotic cells were performed as described 8 using iC3b-opsonized apoptotic thymocytes 12. Non-opsonized interaction between human macrophages and zymosan or LPS (Sigma-Aldrich) was performed without the presence of human serum. Zymosan, at 100 μg/mL, Fluorouracil was added to macrophages for 1 h at 37°C/5% CO2. Macrophages were then washed three times with RPMI. Following the interaction, macrophages were washed three times using ice cold RPMI, followed by incubation for 24 h with RPMI medium, 10% serum. Supernatants were collected at 6 and 24 h. In mixed assays, macrophages or DC were washed three times with RPMI and then exposed for 1 h to either iC3b-opsonized

apoptotic cells or to RPMI. After 1 h, macrophages Selleck C59 wnt were washed three times with RPMI, while DC were not washed; both cell types were then exposed to zymosan 100 μg/mL for 1 h/37°C, and washed three times with RPMI. All macrophages were then incubated in RPMI with 10% human serum for up to 24 h. Supernatants were collected at 6 and 24 h. Interaction index was calculated as described previously 15. Cytokine concentrations were determined for IL-1β, Non-specific serine/threonine protein kinase IL-6, IL-10, and TGF-β using ELISA immunoassays, according to instructions provided with each kit. Data were analyzed using a log/log curve fit option from Microsoft Excel software (Microsoft Corporation, Seattle, WA, USA). In

inhibition assays, anti-IL-10 and anti-TGFβ (R&D Systems) were used. Rabbit polyclonal antibody against human phosphorylated IkB (37 Kd) (R&D Systems) was used to detect protein by immunoblotting. A total of 40×106 freshly isolated macrophages were lysed following the indicated treatments, loaded on 14% SDS-PAGE gel, transferred to a PVDF membrane (Millipore, MA, USA), and blocked with 20% skimmed milk in PBST (PBS*1, 0.05–0.1% Tween 20). The membrane was incubated with primary antibody overnight at 4°C, then washed with TBST and incubated for 30 min with 1:10  000 HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibody. Proteins were visualized with the EZ-ECL detection kit (Beit-Haemek Industries). After interaction with iC3b-opsonized apoptotic cells and or zymosan, DC were fixed with 1% PFA in PBS for 15 min at room temperature and washed twice with PBS containing 10% FBS (PBS-FBS). For microscopy, DC were layered on a microscope slide using a Shandon Cytospin centrifuge (Shandon, Pittsburgh, PA, USA) at 600 rpm for 5 min. Cells were then permeabilized for 45 min with 0.